Presentation on theme: "Syagen Technology, Inc. 1411 Warner Avenue Tustin, CA 92780 www.syagen.com APPI-LC/MS Analysis of Acylglycerols Sheng-Suan Cai, Luke Short, and Jack Syage."— Presentation transcript:
Syagen Technology, Inc. 1411 Warner Avenue Tustin, CA 92780 www.syagen.com APPI-LC/MS Analysis of Acylglycerols Sheng-Suan Cai, Luke Short, and Jack Syage Syagen Technology, Inc. Jonathan Curtis Ocean Nutrition Canada
Photoionization Benefits of Photoionization Ionizes wide range of compounds (e.g., non-polars, electronegative cpds, etc.) Predominantly parent ion signal Minimum fragmentation Minimum solvent signal Minimum ion suppression Signal linear with concentration Solvent (S)Analyte (A) Energy [eV] IP A+A+A+A+ S [A-m] + + m Fragmentation
pump to MS ~ ~ ~ ~ ~ ~ ~ LC eluent / injection cone probe VUV lamp APPI Source
Direct APPI vs. Dopant-assisted APPI Direct APPI M + hv M + + e - M + + S MH + + S[-H] Dopant APPI D + hv D + + e - D + + M MH + + D[-H] D + + M M + + D Analyte molecule M is ionized to a molecular radical ion M +. (If analyte ionization potential is below photon energy) In the presence of protic solvents, M + may abstract a hydrogen atom to form MH +. A photoionizable dopant is delivered in large concentration to yield many D + ions. D + ionizes analyte M by proton or electron transfer. This is PI-initiated APCI.
Published APPI Literature Over 1000 APPI sources in users hands since introduction in 2001 Bibliography available on www.syagen.com
Objectives Developed improved method relative to conventional methods GC or GC/MS requires tedious sample prep and analyte derivatization Conventional LC (i.e., with UV or ELSD) lacks sensitivity and specificity Difficulties in analyzing nonpolar lipids by reversed phase LC/MS due to low solubility of analytes in reversed phase solvent systems (i.e., MeOH:H 2 O or CH 3 CN:H 2 O) Normal phase LC/MS may be better choice To investigate the advantage of using APPI over APCI and ESI for analysis of nonpolar lipids by comparing Mass spectra Dynamic linear range Sensitivity
Selected Target Analytes Four individual non-polar lipid standards were tested EPA and EPA methyl ester (fatty acid group) Monoarachidin (saturated monoglyceride, C20:0) Diarachidin (saturated diglyceride, C20:0) Trielaidin (monounsaturated triglyceride, C18:1) Trielaidin EPA S.- S. Cai and J. A. Syage, Anal. Chem. 78, 1191-1199 (2006). S.- S. Cai and J. A. Syage, J. Chromatogr. A, 1110, 15-26 (2006).
EPA Methyl Ester (MW = 316) Mass Spectra APPI and APCI mobile phase was hexane, ESI mobile phase was 1:1 isooctane/IPA without or with 10 mM ammonium formate APCI+ [M+H] + 5.99e5 [M+H] + APPI+ 9.44e5 [M+H] + [M+Na] + ESI+ 1.71e5 ESI+ [M+H] + [ M+NH 4 ] + [M+Na] + 9.36e5
Comparison of APPI, APCI, and ESI Monoarachidin Linearity Plots. Mobile phase: 1:1 isooctane/IPA (APPI & APCI). 10:15:1 isooctane/IPA/water with 15.4 mM sodium acetate (ESI sodium adduct) and 1:1 isooctane:IPA with 10 mM ammonium formate (ESI ammonium adduct).
Peak Smoothness, Area Count and S/N Ratio APPI+ Area=983 S/N Ratio = 138 APCI+ Area = 445 S/N Ratio = 46 ESI+ Area = 1718 S/N Ratio = 35 EPA Methyl Ester [M+H] +, 1000 pg High area count does not necessarily mean high S/N ratio
Comparison of Detection Limits ESI [M+Na] + signal unstable, NaOAc causes source fouling, ESI [M+NH 4 ] + poor linearity, nonlinear or extremely narrow linear range [M+NH 4 ] + Monoarachidin 0 10 20 30 40 APPI+APCI+ESI+ DL (pg) [M+Na] + [M+NH 4 ] + Day1 Day2 ESI Signal Nonlinear ESI Linear up to only 5 ng [M+NH 4 ] + Diarachidin 0 20 40 60 80 100 120 APPI+APCI+ESI+ DL (pg) ESI Linear up to only 10 ng [M+NH 4 ] +
Chemical Structures of TAG Analytes LnLnLn, C18:3/C18:3/C18:3 LLL, C18:2/C18:2/C18:2 OOO, C18:1/C18:1/C18:1 LLO, C18:2/C18:2/C18:1 SSO, C18:0/C18:0/C18:1 SSS, C18:0/C18:0/C18:0
APPI Full Scan Mass Spectra of TAGs [M+Na] + [M-C18:0] + SSS, C18:0/C18:0/C18:0 [M-C18:1] + [M-C18:0] + [M+Na] + SSO, C18:0/C18:0/C18:1 [M-C18:2] + [M-C18:1] + [M+H] + LLO, C18:2/C18:2/C18:1 OOO, C18:1/C18:1/C18:1 [M-C18:1] + [M+H] + [M+Na] + [M-C18:2] + [M+H] + LLL, C18:2/C18:2/C18:2 [M+H] + [M-C18:3] + LnLnLn, C18:3/C18:3/C18:3 As degree of unsaturation increases, [M+H] + intensity increases
Strategies for Establishments of NA-RP Mobile Phases by Gradient Elution Six possible combinations as binary mobile phase: MeOH : IPA, MeOH : CH 2 Cl 2, MeOH : CHCl 3 CH 3 CN : IPA, CH 3 CN : CH 2 Cl 2, CH 3 CN : CHCl 3 MeOH or CH 3 CN IPA or CH 2 Cl 2 or CHCl 3 or …… Mobile Phase A Weak Solvent Strength Strong Solvent Strength Mobile Phase B Poor solubility Good solubility
Nonaqueous RP-LC Separations of TAGs MeOH:IPA, 9:1 for 0.25 min, linear gradient to 4:6 in 4 min and hold CH 3 CN:IPA, 9:1 for 0.25 min, linear gradient to 3:7 in 4 min and hold MeOH:CHCl 3, 9:1 for 0.25 min, linear gradient to 6:4 in 4 min and hold CH 3 CN:CHCl 3, 9:1 for 0.25 min, linear gradient to 5:5 in 4 min and hold MeOH:CH 2 Cl 2, 9:1 for 0.25 min, linear gradient to 6:4 in 4 min and hold CH 3 CN:CH 2 Cl 2, 9:1 for 0.25 min, linear gradient to 5:5 in 4 min and hold LnLnLn LLL LLO OOO SSO SSS Waters ZQ APPI-LC/MS. Gemini C 18 Column, 150 x 2 mm. Mobile phase flow rate 0.2 mL/min, dopant flow rate 0.04 mL/min. 10 ng each. No dopant Dopant acetone
Mobile Phase: MeOH/IPA No Dopant Acetone Toluene Dopants do not enhance overall sensitivity Peak Area S/N Ratio
Mobile Phase: MeOH/CHCl 3 Dopants enhance performance and acetone wins due to lower baseline noise than toluene No dopant Acetone Toluene Peak Area S/N Ratio
Summary and Conclusions Triacylglycerols in free acid and methyl ester forms in standards and in fish oils were studied by LC/MS using APPI, APCI, and ESI APPI and APCI offer comparable linear range (i.e., 4-5 decades) APPI is 2-4x more sensitive than APCI and much more sensitive than ESI w/o mobile phase additives. ESI sensitivity dramatically enhanced by mobile phase modifiers, but at much reduced linear range. Flow injection LODs <10 pg, and overall on-column LODs are 25 – 200 pg for a wide range of solvent conditions Use “APPI-Friendly” solvents such as IPA or MeOH for high sensitivity w/o dopants Use CH 3 CN or CHCl 3 for lower column backpressure and better resolution, but dopants needed Acetone outperforms toluene as a dopant by not increasing and sometimes even suppressing baseline noise We acknowledge partial funding from NIH
Estimated On-Column Limits of Detection Most of LODs fall below 200 pg levels. Estimated from injections of 1 ng/µL mixed standard with 10 µL injection volume. LODs equivalent to the amount at S/N = 3. MeOH/IPA CH3CN/IPA MeOH/CHCl3 CH3CN/CHCl3 MeOH/CH2Cl2 CH3CN/CH2Cl2 No dopant Acetone Acetone Acetone Acetone Acetone